The present invention relates to the purification of plasmids and pertains particularly to an improved method and apparatus for the purification of plasmids and the like.
A great deal of laboratory research is carried out in which recombinant DNA techniques are utilized. Among the research activities carried out are DNA sequencing, DNA restriction mapping, DNA probe generation, construction of other plasmid or related DNA from smaller pieces, RNA transcription from a plasmid template, hybridization blot analysis, transformation into bacterial, yeast or mammalian cells, S1 nuclease mapping, microinjection into embryos, and election microscopy analysis. All of these require substantially pure concentrations of plasmid DNA.
Many techniques and apparatus exist for small scale purification of plasmid DNA. The typical prior art approach to the purification of plasmids involves a series of steps, including a collection of cells grown in liquid culture by centrifugation, separation of the bacterial chromatic (genomic) DNA, and cellular debris from the soluble contents of the bacteria by centrifugation of filtration, and concentration of the plasmid DNA apart from other cellular components by alcohol or isopropanol, absorption to solid media (i.e. ion exchange resin, glass powder, reverse phase chromatography resin, etc.), or salt precipitation. Additional purification steps may be added to these, such as phenol/chloroform extraction, secondary alcohol precipitation, protease or ribonuclease treatment to further purity the plasmid DNA.
Other methods of plasmid purification include the additional steps of the addition of CsCl to supernatnat from the bacterial lysis after removal of the bacterial genomic DNA, followed by ultracentrifugation. The ultracentrifugation results in a CsCl density gradient in which the plasmid DNA forms a sharp band. This band is removed from the gradient and the DNA separated from the CsCl by alcohol precipitation or other suitable means. These procedures are widely used in molecular biology research for plasmid purification, and have been refined to produce plasmid DNA which is suitable for use in virtually any subsequent procedure of molecular biology.
Certain apparatus have been developed for purification of plasmid DNA. One such apparatus is available from a company called Applied Biosystems, and can purify DNA from samples of tissue, blood, bacteria, etc. The apparatus utilizes repeated organic extraction of sample material to release and purify the DNA. Reagents drawn from reservoirs are automatically introduced into sample containing vials in which the aqueous/organic extraction occurs. Either phenol or guanidine isothiocynate can be used by the machine as the organic phase material. After the extraction steps, the DNA is concentrated on chromatography resin, which is held in the upper portion of the extraction vials. The operator then removes the vials from the instrument and elutes the DNA manually from the resin. This apparatus is designed primarily for purification of genomic DNA from mamalian cells, tissue, blood, etc. and does not perform well with bacterial plasmid DNA. The machine has no capability of separating the plasmid from bacterial DNA.
A fully automated machine is available from Autogen, Inc., which is designed to purity plasmid DNA from recombinant bacteria. This machine is essentially an electronically controlled mechanical robot which performs multiple small scale plasmid purifications. The machine utilizes a precision centrifuge, with sets of disposable plastic tubes into which starting bacterial cultures are placed. Robotic pipet holders positioned above the centrifuge introduce and remove fluids from disposable sample tubes during the run, which involves centrifugation of the samples at two different steps or cycles. This machine can purify up to twelve samples of plasmid DNA in less than an hour. However, the machine is extremely expensive for laboratory use.
Other techniques for separation of substance include electrophoresis separation. Exemplary of this approach are the following U.S. Patents:
Strauch, U.S. Pat. No. 3,533,933, granted Oct. 13, 1970, entitled, "Process and Device for the Isolation of Fractions of a Substance Mixture Electrophoretically Separated in a Carrier Gel", discloses a vertical separation column which is filled partially or completely with a carrier gel with an elution chamber at the bottom of the column.
Levy, U.S. Pat. No. 3,616,454, granted Oct. 26, 1971, entitled "Method of an Apparatus for Electrophoretic Separation in a Gel Column", discloses an apparatus wherein a specimen is placed in the upper end of a polyacrylamide gel column of an electrophoresis whose lower end terminates at a receptacle containing an elution solution.
Nerenberg, U.S. Pat. No. 3,640,813, granted Feb. 8, 1972, entitled "Adapter for a Macromolecule Separation Device", discloses a gel medium disposed in a vertical column with an adapter for the lower end of the column containing a gel and channels for fluid ingress to the upper gel surface and egress therefrom.
The following patents are of interest in disclosing related methods and apparatus:
U.S. Pat. No. 3,579,433, granted May 18, 1971; PA0 U.S. Pat. No. 3,715,295, granted Feb. 6, 1973; PA0 U.S. Pat. No. 3,755,121, granted Aug. 28, 1973; PA0 U.S. Pat. No. 3,951,776, granted Apr. 20, 1976; and PA0 U.S. Pat. No. 4,164,464, granted Aug. 14, 1979.
Many of these existing methods and apparatus have a number of drawbacks, and are generally unsatisfactory in that they are expensive and require many complicated steps and procedures. Others are unable to produce satisfactory purity and quantities.
It is desirable that a simple, inexpensive, yet reliable method and apparatus be available for purification of plasmid DNA starting directly from bacterial culture or collected bacterial cells.